The stimulatory and inhibitory guanine nucleotide regulatory proteins and (N/s and N/i) have recently been identified as pivotal components of hormonally-responsive adenylate cyclase systems. Preliminary experiments in my laboratory suggest that serine protease inhibitors block hormonal activation of cyclase activity by interaction with these N-proteins. These findings open the way for a number of new and potentially exciting investigations regarding the dynamics of gonadotropin receptor-adenylate cyclase coupling. I propose to study the mechanisms of gonadotropin- induced transmembrane signalling as follows: 1. N/s and N/i will be purified from rat liver and thoroughly studied for their ability to interact with isotoptically and fluorescently labeled protease inhibitors. To aid in purification and identification of N- proteins, polyclonal antibodies that are specific for the individual subunits of Ns and Ni will be produced. Synthetic peptides that correspond to defined and unique regions of each subunit will be used as antigens. The effects of protease inhibitors on nucleotide binding and dissociation will be assessed in order to.determine their mode of inhibition of cyclase activity. 2. To confirm interaction of protease inhibitors with membrane bound N-proteins, rat luteal membrane proteins that bind labeled inhibitors will be isolated and identified. If labeled proteins other than Ns and Ni are found, these will be characterized for their influence on adenylate cyclase activity. 3. The LH/hCG receptor will be further purified. Receptor coupling to N-proteins will be investigated by Western blotting using N-protein antibodies and covalent cross-linking using bifunctional reagents. 4. If time permits, fluorescent guanosine 5-triphosphate analogs will be characterized for their effects on rat luteal adenylate cyclase and their ability to bind and activate N-proteins. Expansions of this project will be directed toward development of fluorescence tech- niques to study the dynamic coupling of gonadotropin receptor:N- protein:adenylate cyclase in membranes. These studies should provide a more comprehensive framework for understanding signal transduction in gonadotropin-responsive tissues.